Placental tissue grafts

ABSTRACT

A method for preparing placenta membrane tissue grafts for medical use, includes obtaining a placenta from a subject, cleaning the placenta, separating the chorion tissue from the amniotic membrane, mounting a selected layer of either the chorion tissue or the amniotic membrane onto a drying fixture, dehydrating the selected layer on the drying fixture, and cutting the selected layer into a plurality of tissue grafts. Preferably, the drying fixture includes grooves or raised edges that define the outer contours of each desired tissue graft, after they are cut, and further includes raised or indented logos that emboss the middle area of the tissue grafts during dehydration and that enables an end user to distinguish the top from the bottom side of the graft. The grafts are comprised of single layers of amnion or chorion, multiple layers of amnion or chorion, or multiple layers of a combination of amnion and chorion.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.13/569,095, filed on Aug. 7, 2012, entitled “Methods for Determining theOrientation of a Tissue Graft”, which is a continuation of U.S. patentapplication Ser. No. 11/840,728, filed on Aug. 17, 2007, now U.S. Pat.No. 8,372,437, which claims the benefit under 35 U.S.C. §119(e) of U.S.provisional patent application No. 60/838,467, entitled “Method andSystem for Preserving Amnion Tissue For Later Transplant,” filed Aug.17, 2006. The contents of these applications are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to tissue allografts and, inparticular, to placental membrane tissue grafts (amnion and chorion) andmethods of preparing, preserving, and medical uses for the same.

BACKGROUND OF THE INVENTION

Human placental membrane (e.g. amniotic membrane or tissue) has beenused for various types of reconstructive surgical procedures since theearly 1900s. The membrane serves as a substrate material, more commonlyreferred to as a biological dressing or patch graft. Such membrane hasalso been used widely for ophthalmic procedures in the United States andin countries in the southern hemisphere. Typically, such membrane iseither frozen or dried for preservation and storage until needed forsurgery.

Such placental tissue is typically harvested after an elective Cesareansurgery. The placenta has two primary layers of tissue includingamniotic membrane and chorion. The amniotic membrane is a non-vasculartissue that is the innermost layer of the placenta, and consists of asingle layer, which is attached to a basement membrane. Histologicalevaluation indicates that the membrane layers of the amniotic membraneconsist of epithelium cells, thin reticular fibers (basement membrane),a thick compact layer, and fibroblast layer. The fibrous layer of amnion(i.e., the basement membrane) contains cell anchoring collagen types IV,V, and VII. The chorion is also considered as part of the fetalmembrane; however, the amniotic layer and chorion layer are separate andseparable entities.

Amniotic membrane and chorion tissue provide unique graftingcharacteristics when used for surgical procedures, including providing amatrix for cellular migration/proliferation, providing a naturalbiological barrier, are non-immunogenic, promote increased self-healing,are susceptible of being fixed in place using different techniquesincluding fibrin glue or suturing. And, such grafts, when properlyprepared, can be stored at room temperature for extended periods oftime, without need for refrigeration or freezing, until needed for asurgical procedure.

Known clinical procedures or applications for such amnion grafts includeSchnciderian Membrane repair (i.e. sinus lift), guided tissueregeneration (GTR), general wound care, and primary closure membrane.Known clinical procedures or applications for such chorion graftsinclude biological would dressing.

A detailed look at the history and procedure for harvesting and using“live” amniotic tissue for surgical procedures and a method forharvesting and freezing amniotic tissue grafts for ophthalmic proceduresis described in U.S. Pat. No. 6,152,142 issued to Tseng, which isincorporated herein by reference in its entirety.

There is a need for improved procedures for harvesting, processing, andpreparing amnion and/or chorion tissue for later surgical graftingprocedures.

There is a need for improved procedures for processing and preparingmultiple layers of amnion and/or chorion tissue for later surgicalgrafting procedures.

There is a need for preparing and storing such tissue such that thestroma and basement sides of the tissue are easily and quicklyidentifiable by a surgeon when using such tissue in a surgicalprocedure.

For these and many other reasons, there is a general need for a methodfor preparing placenta membrane tissue grafts for medical use, and thatincludes the steps of obtaining a placenta from a subject, cleaning theplacenta, separating the chorion from the amniotic membrane,disinfecting the chorion and/or amniotic membrane, mounting a selectedlayer of either the chorion or the amniotic membrane onto a dryingfixture, dehydrating the selected layer on the drying fixture, andcutting the selected layer into a plurality of tissue grafts. There isan additional need for a drying fixture that includes grooves or raisededges that define the outer contours of each desired tissue graft andthat make cutting of the grafts more accurate and easy. There is afurther need for a drying fixture that includes raised or indentedlogos, textures, designs, or text that emboss the middle area of thetissue grafts during dehydration and that enables an end user to be baleto distinguish the top surface from the bottom surface of the graft,which is often necessary to know prior to using such grafts in a medicalapplication or surgical procedure. Such logos, textures, designs, ortext can be used for informational purposes or they can, additionallyand advantageously, be used for marketing or advertising purposes. Thereis a need for grafts that are comprised of single layers of amnion orchorion, multiple layers of amnion or chorion, or multiple layers of acombination of amnion and chorion.

The present invention meets one or more of the above-referenced needs asdescribed herein in greater detail.

SUMMARY OF THE INVENTION

One embodiment of the present invention is directed to one or moremethods of preparing placenta membrane tissue grafts, comprising thesteps of obtaining a placenta from a subject, wherein the placentaincludes an amniotic membrane layer and a chorion tissue layer, cleaningthe placenta in a solution, separating the chorion tissue layer from theamniotic membrane layer, mounting a selected layer of either the choriontissue layer or the amniotic membrane layer onto a surface of the dryingfixture, dehydrating the selected layer on the drying fixture, andthereafter, cutting the selected layer into a plurality of placentamembrane tissue grafts. The placenta membrane tissue grafts can beeither amniotic membrane tissue grafts or chorion tissue grafts. Sinceamniotic membrane has a stromal side and an opposite, basement side,when dehydrating an amniotic membrane layer, such layer is mounted ontothe drying fixture with the basement side facing down and stromal sidefacing up.

Preferably, the drying fixture includes a texture or design adapted toemboss such texture or design into the placenta membrane tissue graftsduring the step of dehydration wherein the texture or design embossedinto the placenta membrane tissue enable a user to identify a top andbottom surface of the placenta membrane tissue.

Preferably, the placenta is cleaned in a hyperisotonic solution whereinthe hyperisotonic solution comprises NaCl concentration in a range offrom about 30% to about 10%.

In some embodiments, the method further comprises the step of, afterseparation of the chorion tissue layer from the amniotic membrane layer,soaking the selected layer in an antibiotic solution. Optionally, themethod then also includes the step of rinsing the selected layer toremove the antibiotic solution.

In some embodiments, the method further includes the step of, afterseparation of the chorion tissue layer from the amniotic membrane layer,physically cleaning the selected layer to remove blood clots and othercontaminates.

In other features, the step of dehydrating the selected layer furthercomprises placing the drying fixture in a breathable bag and heating thebag for a predetermined period of time. Preferably, the bag is heated ata temperature of between 35 degrees and 50 degrees Celsius and thepredetermined period of time is between 30 and 120 minutes, wherein 45degrees Celsius and 45 minutes of time in a non-vacuum over or incubatorfor a single layer of tissue generally seems ideal.

In one arrangement, the surface of the drying fixture has a plurality ofgrooves that defines the outer contours of each of the plurality ofplacenta membrane tissue grafts and wherein the step of cuttingcomprises cutting the selected layer along the grooves.

In another arrangement, the surface of the drying fixture has aplurality of raised edges that define the outer contours of each of theplurality of placenta membrane tissue grafts and wherein the step ofcutting comprises rolling a roller across the top of the selected layerand pressing the selected layer against the raised edges.

In another feature, the method further comprises the step of mountingone or more additional layers of chorion tissue or amniotic layer ontothe surface of the drying fixture prior to the step of dehydration tocreate a plurality of laminated placenta membrane tissue grafts having athickness and strength greater than a single layer of placenta membranetissue grafts.

In a further feature, each of the plurality of placenta membrane tissuegrafts is rehydrated prior to use of the respective graft for a medicalprocedure.

In yet further features, the present invention includes tissue graftsprocessed and prepared according to any of the methods described herein.

In another embodiment, the present invention is directed to a tissuegraft that comprises a dehydrated, placenta tissue having a top andbottom surface and an outer contour sized and shaped for use in asuitable medical procedure, wherein a texture or design is embossedwithin the dehydrated, placenta tissue and wherein the embossmentdistinguishes the top from the bottom surface of the placenta tissue;and wherein the dehydrated, placenta tissue graft is usable in thesuitable medical procedure after being rehydrated. In a feature of thisembodiment, the dehydrated, placenta tissue comprises either an amnioticmembrane layer or a chorion tissue layer. In yet another feature, thedehydrated, placenta tissue comprises two or more layers of amnioticmembrane and chorion tissue, wherein the two or more layers include aplurality of amniotic membrane, a plurality of chorion tissue, or aplurality of amniotic membrane and chorion tissue.

These and other aspects of the present invention will become apparentfrom the following description of the preferred embodiment taken inconjunction with the following drawings, although variations andmodifications therein may be affected without departing from the spiritand scope of the novel concepts of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

Further features and benefits of the present invention will be apparentfrom a detailed description of preferred embodiments thereof taken inconjunction with the following drawings, wherein similar elements arereferred to with similar reference numbers, and wherein:

FIG. 1 is a high level flow chart of the primary steps performed in apreferred embodiment of the present invention;

FIG. 2 is an exemplary tissue check-in form used with the preferredembodiment of the present invention;

FIG. 3 is an exemplary raw tissue assessment form used with thepreferred embodiment of the present invention;

FIG. 4 is an exemplary dehydration process form used with the preferredembodiment of the present invention;

FIG. 5 is a perspective view of an exemplary drying fixture for use witha preferred embodiment of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is more particularly described in the followingexamples and embodiments that are intended as illustrative only sincenumerous modifications and variations therein will be apparent to thoseskilled in the art. Various embodiments of the invention are nowdescribed in greater detail. As used in the description herein andthroughout the claims that follow, the meaning of “a”, “an”, and “the”includes plural reference unless the context clearly dictates otherwise.Also, as used in the description herein and throughout the claims thatfollow, the meaning of “in” includes “in” and “on” unless the contextclearly dictates otherwise. Moreover, titles or subtitles may be used inthe specification for the convenience of a reader, which are notintended to influence the scope of the present invention. Additionally,some terms used in this specification are more specifically definedbelow.

Without intent to limit the scope of the invention, exemplaryinstruments, apparatus, methods and their related results according tothe embodiments of the present invention are given below. Note thattitles or subtitles may be used in the discussion of exemplaryembodiments of the present invention for convenience of a reader, whichin no way should limit the scope of the invention. Moreover, certaintheories are proposed and disclosed herein; however, in no way they,whether they are right or wrong, should limit the scope of the inventionso long as the invention is practiced according to the invention withoutregard for any particular theory or scheme of action.

Overview of the Process

Turning first to FIG. 1, a high level overview 100 of the stepsundertaken to harvest, process, and prepare placental material for lateruse as an allograft is disclosed. More detailed descriptions anddiscussion regarding each individual step will follow. At a high level,initially, the placenta tissue is collected from a consenting patientfollowing an elective Cesarean surgery (step 110). The material ispreserved and transported in conventional tissue preservation manner toa suitable processing location or facility for check-in and evaluation(step 120). Gross processing, handling, and separation of the tissuelayers then takes place (step 130). Acceptable tissue is thendecontaminated (step 140), dehydrated (step 150), cut and packaged (step160), and released (step 170) to the market for use by surgeons andother medical professionals in appropriate surgical procedures and forwound care.

Initial Tissue Collection (Step 110)

The recovery of placenta tissue originates in a hospital, where it iscollected during a Cesarean section birth. The donor, referring to themother who is about to give birth, voluntarily submits to acomprehensive screening process designed to provide the safest tissuepossible for transplantation. The screening process preferably tests forantibodies to the human immunodeficiency virus type 1 and type 2(anti-HIV-1 and anti-HIV-2), hepatitis B surface antigens (HBsAg),antibodies to the hepatitis C virus (anti-HCV), antibodies to the humanT-lymphotropic virus type I and type II (anti-HTLV-I and anti-HTLV-II),CMV, and syphilis, using conventional serological tests. The above listof tests is exemplary only, as more, fewer, or different tests may bedesired or necessary over time or based upon the intended use of thegrafts, as will be appreciated by those skilled in the art.

Based upon a review of the donor's information and screening testresults, the donor will either be deemed acceptable or not. In addition,at the time of delivery, cultures are taken to determine the presenceof, for example, Clostridium or Streptococcus. If the donor'sinformation, screening tests, and the delivery cultures are all negative(i.e., do not indicate any risks or indicate acceptable level of risk),the donor is approved and the tissue specimen is designated as initiallyeligible for further processing and evaluation.

Human placentas that meet the above selection criteria are preferablybagged in a saline solution in a sterile shipment bag and stored in acontainer of wet ice for shipment to a processing location or laboratoryfor further processing.

If the placenta tissue is collected prior to the completion or obtainingof results from the screening tests and delivery cultures, such tissueis labeled and kept in quarantine. The tissue is approved for furtherprocessing only after the required screening assessments and deliverycultures, which declare the tissue safe for handling and use, aresatisfied.

Material Check-In and Evaluation (Step 120)

Upon arrival at the processing center or laboratory, the shipment isopened and verified that the sterile shipment bag/container is stillsealed and intact, that ice or other coolant is present and that thecontents are cool, that the appropriate donor paperwork is present, andthat the donor number on the paperwork matches the number on the sterileshipment bag containing the tissue. The sterile shipment bag containingthe tissue is then stored in a refrigerator until ready for furtherprocessing. All appropriate forms, including a tissue check-in form,such as that shown in FIG. 2, are completed and chain of custody andhandling logs (not shown) are also completed.

Gross Tissue Processing (Step 130)

When the tissue is ready to be processed further, the sterile suppliesnecessary for processing the placenta tissue further are assembled in astaging area in a controlled environment and are prepared forintroduction into a critical environment. If the critical environment isa manufacturing hood, the sterile supplies are opened and placed intothe hood using conventional sterile technique. If the criticalenvironment is a clean room, the sterile supplies are opened and placedon a cart covered by a sterile drape. All the work surfaces are coveredby a piece of sterile drape using conventional sterile techniques, andthe sterile supplies and the processing equipments are placed on to thesterile drape, again using conventional sterile technique.

Processing equipment is decontaminated according to conventional andindustry-approved decontamination procedures and then introduced intothe critical environment. The equipment is strategically placed withinthe critical environment to minimize the chance for the equipment tocome in proximity to or is inadvertently contaminated by the tissuespecimen.

Next, the placenta is removed from the sterile shipment bag andtransferred aseptically to a sterile processing basin within thecritical environment. The sterile basin contains, preferably, 18% NaCl(hyperisotonic saline) solution that is at room or near roomtemperature. The placenta is gently massaged to help separate bloodclots and to allow the placenta tissue to reach room temperature, whichwill make the separation of the amnion and chorion layers from eachother, as discussed hereinafter, easier. After having warmed up to theambient temperature (after about 10-30 minutes), the placenta is thenremoved from the sterile processing basin and laid flat on a processingtray with the amniotic membrane layer facing down for inspection.

The placenta tissue is examined and the results of the examination aredocumented on a “Raw Tissue Assessment Form” similar to that shown inFIG. 3. The placenta tissue is examined for discoloration, debris orother contamination, odor, and signs of damage. The size of the tissueis also noted. A determination is made, at this point, as to whether thetissue is acceptable for further processing.

Next, if the placenta tissue is deemed acceptable for furtherprocessing, the amnion and chorion layers of the placenta tissue arethen carefully separated. The materials and equipments used in thisprocedure include the processing tray, 18% saline solution, sterile 4×4sponges, and two sterile Nalgene jars. The placenta tissue is thenclosely examined to find an area (typically a corner) in which theamniotic membrane layer can be separated from the chorion layer. Theamniotic membrane appears as a thin, opaque layer on the chorion.

With the placenta tissue in the processing tray with the amnioticmembrane layer facing down, the chorion layer is gently lifted off theamniotic membrane layer in a slow, continuous motion, using care toprevent tearing of the amniotic membrane. If a tear starts, it isgenerally advisable to restart the separation process from a differentlocation to minimize tearing of either layer of tissue. If the chorionlayer is not needed, it may be gently scrubbed away from the amnioticmembrane layer with one of the sterile 4×4 sponges by gently scrubbingthe chorion in one direction. A new, sterile 4×4 sponge can be usedwhenever the prior sponge becomes too moist or laden with the choriontissue. If the chorion is to be retained, then the separation processcontinues by hand, without the use of the sponges, being careful not totear either the amnion layer or the chorion layer.

Care is then taken to remove blood clots and other extraneous tissuefrom each layer of tissue until the amniotic membrane tissue and thechorion are clean and ready for further processing. More specifically,the amnion and chorion tissues are placed on the processing tray andblood clots are carefully removed using a blunt instrument, a finger, ora sterile non-particulating gauze, by gently rubbing the blood until itis free from the stromal tissue of the amnion and from the trophoblasttissue of the chorion. The stromal layer of the amnion is the side ofthe amniotic membrane that faces the mother. In contrast, the basementmembrane layer is the side of the amnion that faces the baby.

Using a blunt instrument, a cell scraper or sterile gauze, any residualdebris or contamination is also removed. This step must be done withadequate care, again, so as not to tear the amnion or chorion tissues.The cleaning of the amnion is complete once the amnion tissue is smoothand opaque-white in appearance. If the amnion tissue is cleaned toomuch, the opaque layer can be removed. Any areas of the amnion cleanedtoo aggressively and appear clear will be unacceptable and willultimately be discarded.

Chemical Decontamination (Step 140)

The amniotic membrane tissue is then placed into a sterile Nalgene jarfor the next step of chemical decontamination. If the chorion is to berecovered and processed further, it too is placed in its own sterileNalgene jar for the next step of chemical decontamination. If thechorion is not to be kept or used further, it can be discarded in anappropriate biohazard container.

Next, each Nalgene jar is aseptically filled with 18% saline solutionand sealed (or closed with a top. The jar is then placed on a rockerplatform and agitated for between 30 and 90 minutes, which furthercleans the tissue of contaminants.

If the rocket platform was not in the critical environment (e.g., themanufacturing hood), the Nalgene jar is returned to the critical/sterileenvironment and opened. Using sterile forceps, the tissue is gentlyremoved from the Nalgene jar containing the 18% hyperisotonic salinesolution and placed into an empty Nalgene jar. This empty Nalgene jarwith the tissue is then aseptically filled with a pre-mixed antibioticsolution. Preferably, the premixed antibiotic solution is comprised of acocktail of antibiotics, such as Streptomycin Sulfate and GentamicinSulfate. Other antibiotics, such as Polymixin B Sulfate and Bacitracin,or similar antibiotics now available or available in the future, arealso suitable. Additionally, it is preferred that the antibioticsolution be at room temperature when added so that it does not changethe temperature of or otherwise damage the tissue. This jar or containercontaining the tissue and antibiotics is then sealed or closed andplaced on a rocker platform and agitated for, preferably, between 60 and90 minutes. Such rocking or agitation of the tissue within theantibiotic solution further cleans the tissue of contaminants andbacteria.

Again, if the rocker platform was not in the critical environment (e.g.,the manufacturing hood), the jar or container containing the tissue andantibiotics is then returned to the critical/sterile environment andopened. Using sterile forceps, the tissue is gently removed from the jaror container and placed in a sterile basin containing sterile water ornormal saline (0.9% saline solution). The tissue is allowed to soak inplace in the sterile water/normal saline solution for at least 10 to 15minutes. The tissue may be slightly agitated to facilitate removal ofthe antibiotic solution and any other contaminants from the tissue.After at least 10 to 15 minutes, the tissue is ready to be dehydratedand processed further.

Dehydration (Step 150)

Next, the now-rinsed tissue (whether it be the amniotic membrane orchorion tissue) is ready to be dehydrated. The amniotic membrane islaid, stromal side down, on a suitable drying fixture. The stromal sideof the amniotic membrane is the “tackier” of the two sides of theamniotic membrane. A sterile, cotton tipped applicator may be used todetermine which side of the amniotic tissue is tackier and, hence, thestromal side.

The drying fixture is preferably sized to be large enough to receive thetissue, fully, in laid out, flat fashion. The drying fixture ispreferably made of Teflon or of Delrin, is the brand name for an acetalresin engineering plastic invented and sold by DuPont and which is alsoavailable commercially from Werner Machines, Inc. in Marietta, Ga. Anyother suitable material that is heat and cut resistant, capable of beingformed into an appropriate shape to receive wet tissue and to hold andmaintain textured designs, logos, or text can also be used for thedrying fixture. The tissue must be placed on the drying fixture so thatit completely covers as many “product spaces” (as explained hereinafter)as possible.

In one embodiment, similar to that shown in FIG. 5, the receivingsurface of the drying fixture 500 has grooves 505 that define theproduct spaces 510, which are the desired outer contours of the tissueafter it is cut and of a size and shape that is desired for theapplicable surgical procedure in which the tissue will be used, Forexample, the drying fixture can be laid out so that the grooves are in agrid arrangement. The grids on a single drying fixture may be the sameuniform size or may include multiple sizes that are designed fordifferent surgical applications. Nevertheless, any size and shapearrangement can be used for the drying fixture, as will be appreciatedby those skilled in the art. In another embodiment, instead of havinggrooves to define the product spaces, the drying fixture has raisedridges or blades.

Within the “empty” space between the grooves or ridges, the dryingfixture preferably includes a slightly raised or indented texture in theform of text, logo, name, or similar design 520. This textured text,logo, name, or design can be customized or private labeled dependingupon the company that will be selling the graft or depending upon thedesired attributes requested by the end user (e.g., surgeon). Whendried, the tissue will mold itself around the raised texture or into theindented texture—essentially providing a label within the tissue itself.Preferably, such texture/label can be read or viewed on the tissue inonly one orientation so that, after drying and cutting, an end user(typically, a surgeon) of the dried tissue will be able to tell thestromal side from the basement side of the dried tissue. The reason thisis desired is because, during a surgical procedure, it is desirable toplace the allograft in place, with basement side down or adjacent thenative tissue of the patient receiving the allograft. FIG. 5 illustratesa variety of marks, logos, and text 520 that can be included within theempty spaces 510 of the drying fixture 500. Typically, a single dryingfixture will include the same design or text within all of the emptyspaces; however, FIG. 5 shows, for illustrative purposes, a wide varietyof designs that can be included on such drying fixtures to emboss eachgraft.

In a preferred embodiment, only one layer of tissue is placed on thedrying Fixture. In alternate embodiments, multiple layers of tissue areplaced on the same drying fixture to create a laminate-type allograftmaterial that is thicker and stronger than a single layer of allograftmaterial. The actual number of layers will depend upon the surgical needand procedure with which the allograft is designed to be used.

Once the tissue(s) is placed on the drying fixture, the drying fixtureis placed in a sterile Tyvex (or similar, breathable, heat-resistant,and sealable material) dehydration bag and scaled. Such breathabledehydration bag prevents the tissue from drying too quickly. If multipledrying fixtures are being processed simultaneously, each drying fixtureis either placed in its own Tyvex bag or, alternatively, placed into asuitable mounting frame that is designed to hold multiple drying framesthereon and the entire frame is then placed into a larger, singlesterile Tyvex dehydration bag and sealed.

The Tyvex dehydration bag containing the one or more drying fixtures isthen placed into a non-vacuum oven or incubator that has been preheatedto approximately 35 to 50 degrees Celsius. The Tyvex bag remains in theoven for between 30 and 120 minutes, although approximately 45 minutesat a temperature of approximately 45 degrees Celsius appears to be idealto dry the tissue sufficiently but without over-drying or burning thetissue. The specific temperature and time for any specific oven willneed to be calibrated and adjusted based on other factors includingaltitude, size of the oven, accuracy of the oven temperature, materialused for the drying fixture, number of drying fixtures being driedsimultaneously, whether a single or multiple frames of drying fixturesare dried simultaneously, and the like.

An appropriate Dehydration recordation form, similar to that shown inFIG. 4, is completed at the end of the dehydration process.

Cutting & Packaging (Step 160)

Once the tissue has been adequately dehydrated, the tissue is then readyto be cut into specific product sizes and appropriately packages forstorage and later surgical use. First, the Tyvex bag containing thedehydrated tissue is placed back into the sterile/critical environment,The number of grafts to be produced is estimated based on the size andshape of the tissue on the drying fixture(s). An appropriate number ofpouches, one for each allograft, are then also introduced into thesterile/critical environment. The drying fixture(s) are then removedfrom the Tyvex bag.

If the drying fixture has grooves, then the following procedure isfollowed for cutting the tissue into product sizes. Preferably, if thedrying fixture is configured in a grid pattern, a #20 or similarstraight or rolling blade is used to cut along each groove line inparallel. Then, all lines in the perpendicular direction are cut.

If the drying fixture has raised edges or blades, then the followingprocedure is followed for cutting the tissue into product sizes.Preferably, a sterile roller is used to roll across the drying fixture.Sufficient pressure must be applied so that the dehydrated tissue is cutalong all of the raised blades or edges of the drying fixture.

After cutting, each separate piece or tissue graft is placed in arespective “inner” pouch. The inner pouch, which preferably has a clearside and an opaque side, should be oriented clear side facing up. Thetissue graft is placed in the “inner” pouch so that the texture in theform of text, logo, name, or similar design is facing out through theclear side of the inner pouch and is visible outside of the inner pouch.This process is repeated for each separate graft.

Each tissue graft is then given a final inspection to confirm that thereare no tears or holes, that the product size (as cut) is withinapproximately 1 millimeter (plus or minus) of the specified size forthat particular graft, that there are no noticeable blemishes ordiscoloration of the tissue, and that the textured logo or wording isreadable and viewable through the “inner” pouch.

To the extent possible, oxygen is removed from the inner pouch before itis sealed. The inner pouch can be sealed in any suitable manner;however, a heat seal has shown to be effective. Next, each inner pouchis separately packaged in an “outer” pouch for further protection,storage, and shipment.

It should be noted that the above process does not require freezing ofthe tissue to kill unwanted cells, to decontaminate the tissue, orotherwise to preserve the tissue. The dehydrated allografts are designedto be stored and shipped at room or ambient temperature, without needfor refrigeration or freezing.

Product Release (Step 170)

Before the product is ready for shipment and release to the end user, afinal inspection is made of both the inner and outer pouches. This finalinspection ensure that the allograft contained therein matches theproduct specifications (size, shape, tissue type, tissue thickness (# oflayers), design logo, etc.) identified on the packaging label Eachpackage is inspected for holes, broken seals, burns, tears,contamination, or other physical defects. Each allograft is alsoinspected to confirm uniformity of appearance, including the absence ofspots or discoloration.

Appropriate labeling and chain of custody is observed throughout all ofthe above processes, in accordance with accepted industry standards andpractice. Appropriate clean room and sterile working conditions aremaintained and used, to the extent possible, throughout the aboveprocesses.

Overview of Clinical Applications

In practice, it has been determined that the above allograft materialscan be stored in room temperature conditions safely for at least five(5) years.

When ready for use, such allografts are re-hydrated by soaking them inBSS (buffered saline solution), 0.9% saline solution, or sterile waterfor 30-90 seconds.

Amnion membrane has the following properties and has been shown to besuitable for the following surgical procedures and indications: GuidedTissue Regeneration (GTR), Schneiderian Membrane repair, primaryclosure, and general wound care.

Laminated amnion membrane has the following properties and has beenshown to be suitable for the following surgical procedures andindications: GTR, Reconstructive, General Wound Care, Neurological, ENT.

Chorion tissue grafts have the following properties and have been shownto be suitable for the following surgical procedures and indications:Biological Dressing or Covering.

Laminated chorion tissue grafts have the following properties and havebeen shown to be suitable for the following surgical procedures andindications: GTR, Reconstructive, General Wound Care, Neurological, ENT.

Laminated amnion and chorion combined tissue grafts have the followingproperties and have been shown to be suitable for the following surgicalprocedures and indications: Advanced Ocular Defects, Reconstructive,General Wound Care, Biological Dressing.

Although the above processes have been described specifically inassociation with amnion membrane and chorion recovered from placentatissue, it should be understood that the above techniques and proceduresare susceptible and usable for many other types of human and animaltissues. In addition, although the above procedures and tissues havebeen described for use with allograft tissues, such procedures andtechniques are likewise suitable and usable for xenograft and isograftapplications.

In view of the foregoing detailed description of preferred embodimentsof the present invention, it readily will be understood by those personsskilled in the art that the present invention is susceptible to broadutility and application. While various aspects have been described inthe context of screen shots, additional aspects, features, andmethodologies of the present invention will be readily discernabletherefrom. Many embodiments and adaptations of the present inventionother than those herein described, as well as many variations,modifications, and equivalent arrangements and methodologies, will beapparent from or reasonably suggested by the present invention and theforegoing description thereof, without departing from the substance orscope of the present invention. Furthermore, any sequence(s) and/ortemporal order of steps of various processes described and claimedherein are those considered to be the best mode contemplated forcarrying out the present invention. It should also be understood that,although steps of various processes may be shown and described as beingin a preferred sequence or temporal order, the steps of any suchprocesses are not limited to being carried out in any particularsequence or order, absent a specific indication of such to achieve aparticular intended result. In most cases, the steps of such processesmay be carried out in various different sequences and orders, whilestill falling within the scope of the present inventions. In addition,some steps may be carried out simultaneously. Accordingly, while thepresent invention has been described herein in detail in relation topreferred embodiments, it is to be understood that this disclosure isonly illustrative and exemplary of the present invention and is mademerely for purposes of providing a full and enabling disclosure of theinvention. The foregoing disclosure is not intended nor is to beconstrued to limit the present invention or otherwise to exclude anysuch other embodiments, adaptations, variations, modifications andequivalent arrangements, the present invention being limited only by theclaims appended hereto and the equivalents thereof.

What is claimed is:
 1. A dehydrated, laminated tissue graft consistingessentially of one or more washed and/or substantially cleaned amnionlayers and one or more washed and/or substantially cleaned chorionlayers, wherein at least one of the amnion layers contains itsfibroblast cell layer, and further wherein the amnion layer and thechorion layer are directly laminated to each other.
 2. The dehydrated,laminated tissue graft of claim 1, wherein said assembled and laminatedlayers are dehydrated together in a drying fixture.
 3. The dehydrated,laminated tissue graft of claim 1, wherein one or both of the one ormore amnion layers and the one or more chorion layers are treated withan antibiotic prior to lamination.
 4. The dehydrated, laminated tissuegraft of claim 1, wherein the tissue graft has been exposed tosterilizing conditions.
 5. The dehydrated, laminated tissue graft ofclaim 1, consisting essentially of one amnion layer and one chorionlayer.
 6. A dehydrated, laminated tissue graft, wherein the tissue graftconsists essentially of one or more amnion layers and one or morechorion layers wherein each of the amnion and chorion layers: areseparated from a placenta to provide separate layers of amnion andchorion, washed and substantially cleaned without removal of thefibroblast cell layer from the amnion layer, and are layered directlyover each other and are laminated and heat dehydrated together toprovide the dehydrated, laminated tissue graft.
 7. The tissue graft ofclaim 6, consisting essentially of one amnion layer and one chorionlayer.
 8. The tissue graft of claim 6, wherein at least one of theamnion or chorion layers has been chemically decontaminated.
 9. Adehydrated, laminated, placental tissue graft which is a laminatecomprising two or more separated and washed layers which layers areselected from amnion and/or chorion wherein the layers are directlylaminated to each other and at least one of said layers is an amnionlayer which retains an epithelial cellular layer.
 10. A dehydrated,laminated, placental tissue graft which is a laminate comprising two ormore separated and washed layers which layers are selected from amnionand/or chorion wherein the layers are directly laminated to each otherand at least one of said layers is an amnion layer which retains afibroblast cellular layer.